The invention relates to compositions and methods of treating a mammal suffering from a disease, disorder or a condition associated with the central nervous system, using isolated stromal cells.
Neurological damage in a mammal having as its genesis trauma, tumor formation or a genetic or other component, is very difficult to treat and/or reverse in the mammal. One treatment for neurological damage to the central nervous system is neurotransplantation. Over the last few decades, neurotransplantation has been used to explore the development, plasticity, and regeneration of the central nervous system (McKay, 1997, Science 276:66-71). Also, neurotransplantation has been used to effect the repair and functional restoration of diseased and damaged nervous tissues (Bjorklund, 1993, Nature 362:414-415; Olson, 1997, Nature Med. 3:1329-1335; Spencer et al., 1992, N. Engl. J. Med. 327:1541-1548: Freed et al., 1992, N. Engl. J. Med 327:1549-1555; Kordower et al., 1995, N. Engl. J. Med. 332:1118-1124; Defer et al., 1996, Brain 119:41-50; Lopez-Lozano et al., 1997, Transp. Proc. 29:977-980; Rosenstein, 1995, Exp. Neurol. 33:106; Turner et al., 1993, Neurosurg. 33:1031-1037; Kang et al., 1993, J. Neurosci. 13:5203-5211; Andersson et al., 1993, Int. J. Dev. Neurosci. 11:555-568; Sanberg et al., 1997, Nature Med. 3:1129-1132). In particular, a series of human patients with Parkinson""s disease have been treated by neurotransplantation of mesencephalic cells obtained from 6 to 9 week old abortuses of human fetuses (Spencer et al., 1992, N. Engl. J. Med. 327:1541-1548: Freed et al., 1992, N. Engl. J. Med 327:1549-1555; Kordower et al., 1995, N. Engl. J. Med. 332:1118-1124; Defer et al., 1996, Brain 119:41-50; Lopez-Lozano et al., 1997, Transp. Proc. 29:977-980). Some of the patients exhibited significant improvement both in clinical symptoms and in the synthesis of dopamine, as assessed by fluorodopa uptake using positron-emission tomography (Spencer et al., 1992, N. Engl. J. Med. 327:1541-1548: Freed et al., 1992, N. Engl. J. Med 327:1549-1555; Kordower et al., 1995, N. Engl. J. Med. 332:1118-1124; Defer et al., 1996, Brain 119:41-50). However, the process of obtaining fetal tissue for therapeutic uses has presented major logistic and ethical barriers (Rosenstein, 1995, Exp. Neurol. 33:106; Turner et al., 1993, Neurosurg. 33:1031-1037). Also, only about 5 to 10% of dopaminergic neurons survive, apparently because of adverse immune reaction to the same (Lopez-Lozano et al., 1997, Transp. Proc. 29:977-980) and because the fetal tissue is primarily dependent on lipid instead glycolytic metabolism (Rosenstein, 1995, Exp. Neurol. 33:106). For these reasons, attempts have been made to develop alternative cells such as fibroblasts (Kang et al., 1993, J. Neurosci. 13:5203-5211), fetal astrocytes (Andersson et al., 1993, Int. J. Dev. Neurosci. 11:555-568), and sertoli cells (Sanberg et al., 1997, Nature Med. 3:1129-1132) which are suitable for neurotransplantation.
In order to treat diseases, disorders, or conditions of the central nervous system, such as for example brain tumors, brain trauma, Huntington""s disease, Alzheimer""s disease, Parkinson""s disease, and spinal cord injury, by transplantation, donor cells should be easily available, capable of rapid expansion in culture, immunologically inert, capable of long term survival and integration in the host brain tissue, and amenable to stable transfection and long-term expression of exogenous genes (Bjorklund, 1993, Nature 362:414-415; Olson, 1997, Nature Med. 3:1329-1335). Donor cells meeting these criteria are not currently available.
In addition to the hematopoietic stem cells, bone marrow contains stem-like precursors for non-hematopoietic cells, such as osteoblasts, chondrocytes, adipocytes and myoblasts (Owen et al., 1988, in Cell and Molecular Biology of Vertebrate Hard Tissues, Ciba Foundation Symposium 136, Chichester, UK, pp. 42-60; Caplan, 1991, J. Orthop. Res. 9:641-650; Prockop, 1997, Science 276:71-74). Non-hematopoietic precursors of the bone marrow have been variously referred to as colony-forming-unit-fibroblasts, mesenchymal stem cells, and marrow stromal cells (MSCs).
MSCs are mesenchymal precursor cells (Friedenstein et al., 1976, Exp. Hemat. 4:267-274) that are characterized by their adherence properties when bone marrow cells are removed from a mammal and are transferred to plastic dishes. Within about four hours, MSCs adhere to the plastic and can thus be isolated by removing non-adherent cells from the dishes. Bone marrow cells, i.e., MSCs, that tightly adhere to plastic have been studied extensively (Castro-Malaspina et al., 1980, Blood 56:289-30125; Piersma et al., 1985, Exp. Hematol 13:237-243; Simmons et al., 1991, Blood 78:55-62; Beresf ord et al., 1992, J. Cell. Sci. 102:341-3 51; Liesveld et al., 1989, Blood 73:1794-1800; Liesveld et al., Exp. Hematol 19:63-70; Bennett et al., 1991, J. Cell. Sci. 99:131-139). The terms xe2x80x9cMSCsxe2x80x9d and xe2x80x9cstromal cellsxe2x80x9d are used interchangeably herein.
Stromal cells are believed to participate in the creation of the microenvironment within the bone marrow in vivo. When isolated, stromal cells are initially quiescent but eventually begin dividing so that they can be cultured in vitro. Expanded numbers of stromal cells can be established and maintained. Stromal cells have been used to generate colonies of fibroblastic adipocytic and osteogenic cells when cultured under appropriate conditions. They can also be made to differentiate into cartilage cells and myoblasts. If the adherent cells are cultured in the presence of hydrocortisone or other selective conditions, populations enriched for hematopoietic precursors or osteogenic cells are obtained (Carter et al., 1992, Blood 79:356-364 and Bienzle et al., 1994, Proc. Natl. Acad. Sci USA, 91:350-354).
There are several examples of the use of stromal cells for treatment of disease. For example, European Patent EP 0,381,490, discloses gene therapy using stromal cells. In particular, a method of treating hemophilia is disclosed. Stromal cells have been used to produce fibrous tissue, bone or cartilage when implanted into selective tissues in vivo (Ohgushi et al., 1989, Acte. Orthop. Scand. 60:334-339; Nakahara et al., 1992, J. Orthop. Res. 9:465-476; Niedzwiedski et al., 1993, Biomaterials 14:115-121; and Wakitani et al., 1994, J. Bone and Surg. 76A:579-592). In some reports, stromal cells have been used to generate bone or cartilage in vivo when implanted subcutaneously with a porous ceramic (Ohgushi, et al., 1989, Acta. Orthop. Scand. 60:334-339), intraperitoneally in a diffusion chamber (Nakahara et al., 1991, J. Orthop. Res. 9:465-476), percutaneously into a surgically induced bone defect (Niedzwiedski, et al., 1993, Biomaterials 14: 115-121), or transplanted within a collagen gel to repair a surgical defect in a joint cartilage (Wakitani et al., 1994, J. Bone Surg. 76A: 579-592). Piersma et al. (1983, Brit. J. Hematol. 94:285-290) disclose that after intravenous bone marrow transplantation, the fibroblast colony-forming cells which make up the hemopoietic stroma lodge and remain in the host bone marrow. Stewart et al. (1993, Blood 81:2566-2571) recently observed that unusually large and repeated administrations of whole marrow cells produced long-term engraftment of hematopoietic precursors into mice that had not undergone marrow ablation. Also, Bienzle et al. (1994, Proc. Natl. Acad. Sci. USA, 91:350-354) successfully used long-term bone marrow cultures as donor cells to permanently populate hematopoietic cells in dogs without marrow ablation. In some reports, stromal cells were used either as cells that established a microenvironment for the culture of hematopoietic precursors (Anklesaria, 1987, PNAS USA 84:7681-7685) or as a source of an enriched population of hematopoietic stem cells (Kiefer, 1991, Blood 78(10):2577-2582).
Given the paucity of successful treatments for diseases, disorders and conditions of the central nervous system, there remains a need for additional methods of treating patients affected by a disease, disorder, or condition of the central nervous system. The present invention satisfies this need and overcomes the deficiencies of prior art treatments.
The invention relates to a method of treating a human patient having a disease, disorder or condition of the central nervous system. The method comprises obtaining a bone marrow sample from a human donor, isolating stromal cells from the bone marrow sample, and administering the isolated stromal cells to the central nervous system of the human patient, wherein the presence of the isolated stromal cells in the central nervous system effects treatment of the disease, disorder or condition.
In one aspect, the human donor is not suffering from a disease, disorder or condition of the central nervous system and the human donor is synergeneic with the patient.
In another aspect, the human donor is the human patient.
In yet another aspect, the disease, disorder or condition of the central nervous system is selected from the group consisting of a genetic disease, a tumor, trauma and stroke.
In a preferred embodiment, the disease, disorder or condition is injury to the tissues or cells of the central nervous system. In another preferred embodiment, the disease, disorder or condition is a brain tumor.
In another aspect, the isolated stromal cells administered to the central nervous system remain present or replicate in the central nervous system.
In a further aspect, prior to administering the isolated stromal cells, the cells are cultured in vitro.
In yet a further aspect, prior to administering the isolated stromal cells, the isolated stromal cells are transfected with an isolated nucleic acid encoding a therapeutic protein, wherein when the protein is expressed in the cells the protein serves to effect treatment of the disease, disorder or condition.
In a preferred embodiment, the therapeutic protein is selected from the group consisting of a cytokine, a chemokine and a neurotrophin.
In yet another aspect, prior to administering the isolated stromal cells, the isolated stromal cells are transfected with an isolated nucleic acid encoding a therapeutic protein wherein when such protein is secreted by the cells the protein serves to effect treatment of the disease, disorder or condition.
In a preferred embodiment, the isolated nucleic acid is operably linked to a promoter/regulatory sequence. In yet another preferred embodiment, the therapeutic protein is selected from the group consisting of a cytokine, a chemokine and a neurotrophin. And, in a further preferred embodiment, the isolated nucleic acid is a wild type copy of a mutated, non-functioning or under-expressed gene, wherein the isolated nucleic acid is operably linked to a promoter/regulatory sequence and is expressed in the isolated stromal cells.
In yet another preferred embodiment, the isolated nucleic acid is a wild type copy of a mutated, non-functioning or under-expressed gene, wherein the isolated nucleic acid is operably linked to a promoter/regulatory sequence and is expressed in to generate a protein which is secreted from the isolated stromal cells.
In another aspect of this aspect of the invention, prior to administrating the stromal cells, the cells are pre-differentiated by coculturing the stromal cells in the presence of a substantially homogeneous population of differentiated cells, whereby the stromal cells differentiate and acquire the phenotypic characteristics of the differentiated cells.
In yet another aspect, prior to administration of the isolated stromal cells at least one of the steps of culturing the cells in vitro, introducing isolated nucleic acid into the cells, and pre-differentiating the cells, is performed.
In another aspect, the isolated stromal cells are immunologically isolated.
The invention also relates to a method of directing the differentiation of an isolated stromal cell comprising culturing the isolated stromal cell in the presence of a substantially homogeneous population of differentiated cells whereby the isolated stromal cell differentiates and acquires the phenotypic characteristics of the differentiated cells.
In one aspect of this aspect of the invention, the differentiated cells are astrocytes.